Rapid release mini-tablets provide analgesia in laboratory animals

ABSTRACT

Pellets containing an analgesic uniformly dispersed in a lipid carrier such as cholesterol mixed with fatty acid esters, can be used to provide long term pain relief. 5 mg cholesterol-tryglyceride-buprenorphine pellets released the majority of drug in 24-48 hours after implant and provide clinically significant plasma levels of analgesia in mice for 3-9 days. Blood levels of analgesia peak at day-1 and are substantially complete by day-5 depending on the level of buprenorphine. These results demonstrate that post surgical implants provide clinically significant levels of analgesia in the 24-48 hour period following surgery and thus obviate the time consuming, expensive, and high-risk need to inject mice post surgery. The pellets are safe and easy to use. Placed in the surgical wound at the end of surgery, they provide 2-3 days of analgesia and obviate the need for subsequent handling of the animal for pain therapy. The implants have no detectable effect on mouse behavior, hematology, or liver chemistry. The unexpected release kinetics of the 5 mg pellet provides an ideal implant for post surgical analgesia. These implants solve a significant problem facing scientists who use rodents in research and abide by international of animal welfare.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of pending prior application U.S.Ser. No. 11/876,102 filed Oct. 22, 2007, entitled “Rapid ReleaseMini-Tablets Provide Analgesia in Laboratory Animals”, by MichaelGuarnieri, which claims priority to U.S. Ser. No. 60/853,904, filed Oct.24, 2006, both of which are herein incorporated by reference in theirentirety.

FIELD OF THE INVENTION

This is generally in the field of controlled drug delivery, and inparticular is a long term analgesic formulation for implantation inlaboratory animals.

BACKGROUND OF THE INVENTION

Regulations for animal research require humane treatment of laboratoryanimals. Because animals cannot articulate pain, humane principlesmandate that scientists provide the same analgesia that humans need forsimilar surgery. Indeed, an increasing body of evidence has shown thatpain receptors in rodents and humans are virtually identical and thatthe sensation of pain would be highly comparable (Cowan, et al. Eur JPharmacology, 507: 87-98, 2005). A second principal is that regulationsfor animal research be internationally harmonized so that pharmaceuticalstudies have the same scientific basis in every country.

The U.S. Department of Agriculture (USDA) regulates animal research inthe US (Animal Welfare Act and Amendments, (7 U.S.C. 2131 et seq APHIS,USDA). With one exception, USDA regulations for animal welfare aresimilar to regulations in the Europe, Japan, and other industrializednations. In the US, mice and rats are not considered to be “animals.”This exception allows scientists in the US to provide lower and lessexpensive standards of analgesia. Studies have shown that at least 80%of scientists fail to provide adequate post-surgical analgesia forrodents (Richardson, et al. Altern Lab Anim 33: 119-127, 2005). Notsurprisingly, the exception is the basis of anti-USDA lawsuits fromanimal welfare organizations.

It is estimated that more than 60 million mice and rats are used yearlyin medical and toxicology research, with the majority of rodents beingused in the US. The loophole in USDA regulations regarding the postsurgical care of rodents may play a role in these use patterns. Manyobservers predict that US regulations will soon be come harmonized withinternational regulations and that rodents will be afforded greaterprotection based on scientific concerns and humane principles.

A low cost, safe and easy to use analgesic for rodent surgery could meetmany of the concerns that have been articulated by scientists in the US.Scientists have defended their reluctance to use analgesia for rodentsbased on the expense of post-surgical care, concerns that analgesiacould interfere with the outcome of an experiment, and the difficulty ofhandling small animals.

The cost of post-surgical care can be significant and the management ofanalgesia requires expensive labor resources. Drugs such as aspirin andibuprofen must be given orally (PO) at 4-6 hour intervals. Morphine andbuprenorphine have to be injected subcutaneously (SQ) orintraperitonealy (IP) at 6-8 hour intervals. Injured animals do not eator drink on normal schedules, so food and water cannot be effectivelyspiked with drugs. Whether post-surgical analgesia affects the outcomeof an experiment can be determined. Scientists routinely state theirconcerns that affects can occur (Karas A Z, Lab Animal 35: 38-45, 2006).Frequently, these statements are made without experimental proof(Silverman J, Lab Animal, 30 (3): 21-26, 2001). It is highly doubtfulthat among the many choices of analgesic drugs, one or more drug couldnot be found to humanely treat the animal and not interfere with theexperiment.

The difficulty in providing analgesia to small animals is perhaps themost significant barrier to rodent care. It is difficult to grab andhold a mouse and rat without disturbing the surgical wound, injuring thespine, and without causing more pain. Once restrained, animals resist POtherapy. SC and IP injections cause pain. IP injections cause infectionsand death if the needle penetrates sensitive tissue (Coria-Avila, et al.Lab Animal 36: 25-30, 2007.) A chronic delivery form of analgesia forrodent surgery would have significant benefits.

Several efforts have been made to use chronic drug-delivery systems suchas infusion pumps and biodegradable scaffolds for laboratory andcompanion animal medicine. Alzet osmotic pumps and other swellable coretechnologies have become a standard method for delivering constantlevels of drugs to animals for research (Guarnieri, et al. JNeuroscience Methods, 144: 147-152, 2005). Additional efforts haveincluded the use of transdermal patches (Bohme Clin Rheumatol, 21:S13-S16, 2002), and food-based drugs. Transdermal patches have limiteduse because rodents and companion animals scratch and remove skinpatches. Food-based analgesia has not been widely studied becausepost-surgical animals frequently have erratic eating patterns andbecause of the costs associated with adding drugs to standard chows.

An implantable, rapid-release analgesic would significantly reducebarriers to humane post surgical animal care. However, as demonstratedby the examples, most controlled release systems do not provide thenecessary blood levels of analgesics required. Biodegradable drugimplants would eliminate the safety concerns about handling smallanimals after surgery. Cholesterol implants have been used to providesustained release of macromolecules, but not for the short-term deliveryof analgesia (U.S. Pat. No. 4,452,775 to Kent. Lipospheres forcontrolled delivery of substances have been described in U.S. Pat. No.5,188,827 to Domb. Liposomes, solid lipid nanoparticles, oilysuspensions, and fatty acids for sustained release parenteralformulations and implants have been described in U.S. Pat. No. 5,137,874to Cady. Waxes, lipid microspheres, and lipid implants have beendescribed as sustained-release vehicles, as reviewed by Mohl S, TheDevelopment of a Sustained and Controlled Release Device forPharmaceutical Proteins based on Lipid Implants. Dissertation,University of Munich, 2004. In the majority of cases, the lipid andother biodegradable carrier such as PLGA copolymers (Shaw et al, 1993)are designed for the long-term sustained release of drugs and vaccines(U.S. Pat. No. 4,164,560 to Folkman and Langer.

Several exceptions can be noted to the focus of biodegradable lipidscaffolds and long-term drug delivery. Lidocaine and bupivcainebiodegradable scaffolds such as SABER and lipospheres have been designedfor 2-3 hour duration of anesthesia for surgical wounds (Hersh, et al.Anesth Prog. 39(6): 197-200, 1992; Toongsuwan, et al. Int JPharmaceutics 280: 57-65, 2004). Liposomal preparations of morphine andoxymorphone have been tested in mouse and rat pain models (Grant, et al.Anesthesia and Analgesia 79: 706-709, 1994; Krugner-Higby, et al.Comparative Medicine 53: 270-279, 2003; Clark, et al. Comp Med, 54:558-563, 2004). Liposomes, however are difficult to prepare and must beused fresh. Morphine is a more highly regulated drug and provides lessanalgesic efficacy compared to buprenorphine.

Buprenorphine esters can act as buprenorphine pro drugs in sesame,castor, cottonseed, peanut or soybean oil solutions injected into ratmuscle (IM). The analgesic effect for sesame oil solutions could bemeasured for approximately 3 days (Liu, et al. Anesth Analg. 102:1445-1451, 2006; J Pharm Pharmacol. 58(3): 337-34, 2006). The laterstudies contrast with our inability (Table 5) to detect buprenorphine inblood following SC injections of buprenorphine in oleic acid, an oilwith properties highly similar to the oils used by Liu and coworkers.

Pontani, et al., Pharmacology Biochemistry & Behavior 18: 471-474, 1983and Xenobiotica, 15, 287-297, 1985 describe a 50 mg cholesterol implantdesigned for the sustained release of buprenorphine for long-termtherapy to prevent opiate seeking behavior. The 50 mg implant releasesbuprenorphine at a uniform rate (first order kinetics) for at least 12weeks. While this release profile is suitable for sustained delivery ofanti-addiction medication, it is highly unsuitable for short-term postsurgical analgesia. The long-term presence of a foreign body and drugcould have significant impact on research on vaccines, neurodegenerativedisease, cancer, immunity-related chronic diseases such as diabetes, andother diseases.

It is therefore an object of the present invention to provide acontrolled release formulation for delivery of analgesics to laboratoryand research animals, especially small rodents.

SUMMARY OF THE INVENTION

Pellets containing an analgesic uniformly dispersed in a lipid carriersuch as cholesterol mixed with fatty acid esters, can be used to providelong term pain relief. 5 mg cholesterol-triglyceride-buprenorphinepellets released the majority of drug in 24-48 hours after implant andprovide clinically significant plasma levels of analgesia in mice for3-9 days. Blood levels of analgesia peak at day-1 and are substantiallycomplete by day-5 depending on the level of buprenorphine. These resultsdemonstrate that post surgical implants provide clinically significantlevels of analgesia in the 24-48 hour period following surgery and thusobviate the time consuming, expensive, and high-risk need to inject micepost surgery. The pellets are safe and easy to use. Placed in thesurgical wound at the end of surgery, they provide 2-3 days of analgesiaand obviate the need for subsequent handling of the animal for paintherapy. The implants have no detectable effect on mouse behavior,hematology, or liver chemistry. The unexpected release kinetics of the 5mg pellet provides an ideal implant for post surgical analgesia. Theseimplants solve a significant problem facing scientists who use rodentsin research and abide by international of animal welfare.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph of percent remaining drug over time in days comparingthe pharmacokinetics of a prior art 50 mg pellet with a 5 mg pellet asdescribed herein.

DETAILED DESCRIPTION OF THE INVENTION

I. Formulations

A. Analgesic

Any of the opioids such as buprenorphine and butorphanol, or drugs suchas fentanyl can be used. Representative local anesthetics includebupivacaine, ropivacaine, dibucaine, procaine, chloroprocaine,prilocalne, mepivacaine, etidocaine, tetracaine, lidocaine, andxylocalne, and mixtures thereof can also be used, alone or incombination with other analgesics.

In the preferred embodiment, buprenorphine is recommended for moderateto severe acute pain in mice and rats. The recommended dose in miceranges from 0.05-2.0 mg/kg, SC q6-12 hrs (Flecknell, 2000), (Gades,Danneman et al., 2000). Recent studies have shown that the drug has abroad analgesic profile in a wide range of rodent models of acute andchronic pain [Christoph et al, Eur J Pharmacol 507: 87-98; 2005]. Themechanism of action has been studied in mice (Kogel, et al. Eur. J Pain,9: 599-611, 2005; Ozdogan et al, Eur J. Pharmacol. 529:105-113; 2006).However, as with most studies of buprenorphine in rodents, these studieshave aimed at understanding the drug's action in humans as a medicationfor treating heroine addiction.

The effective dosages for drugs other than buprenorphine can becalculated based on relative potencies of the various drugs. See, forexample, EP 1242087 to Rechitt.

B. Pellet Composition

In the preferred embodiment, the formulation contains cholesterol oranother natural or GRAS sterol. The cholesterol is mixed with atriglyceride or fatty acid ester such as glyceryl tristearate.

Other ingredients can be added, such as antibiotics andantiinflammatories. Representative antiinflammatories includeglucocorticosteroids such as dexamethasone, cortisone, prednisone,hydrocortisone, beclomethasone dipropionate, betamethasone, flunisolide,methylprednisone, paramethasone, prednisolone, triamcinolone,alclometasone, amcinonide, clobetasol, fludrocortisone, diflorasonediacetate, fluocinolone acetonide, fluocinonide, fluorometholone,flurandrenolide, halcinonide, medrysone and mometasone andpharmaceutically acceptable mixtures and salts thereof, andnon-steroidal antiinflammatories such as rimadyl, and aspirin.

II. Method of Manufacture

The preferred method of manufacture is described below. Cholesterol andtriglyceride are dissolved in a suitable solvent such as a halogenatedsolvent and alcohol (5:1), chloroform or methylene chloride, preferablychloroform, to form a liposome or liposphere, analgesic is added to thedesired drug loading, the drug and carrier are mixed to form a uniformdispersion, and the solvent removed by evaporation or other techniquesknown to those in the art. The resulting dry powder is then compressedor extruded to form pellets, preferably less than 5 mm in diameter andlength, more preferably 3 mm or less.

In the preferred embodiment, pellets are loaded with 0.1 to 50% byweight drug, more preferably 2-20% by weight drug. In a preferredembodiment, pellets contain approximately 5 mg:0.100 mg drug, to 5mg:1.0 mg drug.

III. Method of Use

The pellets are implanted at the time of surgery, if possible, orimmediately thereafter using a catheter or trocar. The pellets can beimplanted IM, SC, or IP. The amount administered is based on aneffective amount of anesthetic per kg animal weight, as demonstrated bythe following examples. The preferred dosage for mice is between 0.5 and2 mg/kg.

In the preferred embodiment, the animals are rodents such as mice, rats,gerbils, hamsters, rabbits or guinea pigs. Other animals can also betreated, such as dogs, cats, pigs, and livestock such as horses, goats,sheep, cattle and cameloids (lamas, alpaca, vicuna).

The product is sold as single use surgical implant or in a disposableloaded syringe.

The present invention will be further understood by reference to thefollowing non-limiting examples. References cited herein arespecifically incorporated by reference.

Materials and Methods

Animals: 6-8 week old female BalbC mice were purchased from Harlan,provided with Baltimore City water and standard pelleted chow, andmaintained by the Johns Hopkins University Department of ComparativeMedicine, Animal Care facility under a protocol approved by theInstitute Animal Care and Use Committee.

Reagents: Chloroform (J. T. Baker (Hydrocarbon Stabilized) HPLC-gradewas obtained from a Johns Hopkins University supply, ethanol wasobtained from the Johns Hopkins Hospital supply. Solvents were usedfresh. Cholesterol (Min. 99%, Sigma C 8667), glyceryl-tristearate(Approx. 99%, Sigma T 5061), oleic acid, and buprenorphine HCl (Sigma B9275) were purchased from Sigma-Aldrich (St. Louis, Mo. 63178).Certificates of Analysis, Certificates of Origin, and Material SafetyData Sheets for each product are available at Sigma-Aldrich.com.

Pellet Preparation: Pellets were prepared using the technique previouslydescribed for preparing Gliadel wafers (Guarnieri, Cancer Chemotherapyand Pharmacology, 50, 392-396, 2002). The 20% (w:w) drug pellets wereprepared by dissolving 360 mg of cholesterol, 40 mg of glyceryltristearate, and 100 mg of buprenorphine in 50 mL chloroform and 15 mlof ethanol. 2% (w:w) drug pellets were prepared from 450 mg ofcholesterol, 40 mg of glyceryl stearate and 10 mg of buprenorphine in 50mL chloroform and 15 ml of ethanol. Solvent solutions were evaporatedusing a Buchi roto-evaporator at reduced pressure and 37° water bath for24 hours. No effort was made to monitor the evaporator vacuum or tomonitor the water bath temperature.

In all cases, the resulting powders were dry, odorless, and easilytransferred from the round bottomed evaporation flask with a sterilespatula. Using aseptic techniques, the resulting dry powders weretransferred to a 15 mL screw-capped test tube and stored at −20° untilused for pellet making Pellets were pressed by allowing the drug powderto warm to room temperature and measuring 5±0.2 mg aliquots onto sterileweighing paper. The powder was transferred to a hand press having a 3 mmdie. Pellets were pressed using approximately 2 tons of pressure for 15seconds. The resulting 1×3 mm cylindrical pellets were stored at −20°before in vivo or in vitro tests.

Oleic Acid Emulsion: An oleic acid emulsion of drug was prepared byadding 500 mg of oleic acid from a freshly opened vial and 1 mg of drugto a 2 mL Eppendorf tube. A battery powered hand mixer with apolyethylene homogenizer was used for 30-60 seconds to blend the druginto the oil. The resulting emulsion was transferred immediately to a 1mL syringe for injection into mice.

Dissolution Tests: Pellets in a screw capped test tube containing 10 mLof saline were placed in a 37° shaking water bath. At intervalsdescribed in Results, 1 mL aliquots of supernatant were removed andexamined at 285 nm for buprenorphine.

Buprenorphine Measurements: Buprenorphine released into the salinesolutions used for in vitro dissolution tests was measured at 285 nm indisposable cuvets using a standard curve prepared with 1 mg/ml solutionsof buprenorphine in 1% ethanol in water. Buprenorphine in pellet andpowders was measured by crushing the pellet using a glass spatula andmixing the powder in a 15 mL centrifuge tube with 1 mL of petroleumether and 1 mL of 0.5 N HCl solution. The mixtures were vortexed for 15seconds and centrifuged for 10 min at low speed to separate the solventphases. The acidic aqueous phase was re-extracted 4 times for a total of5 extractions to afford a clear aqueous phase which was examined at 285nm for buprenorphine. The organic phases were discarded in laboratorychemical waste bottles for solvent recovery and recycling. Buprenorphinein plasma was measured by ELISA assay (Cirimele, et al. Forensic ScienceInternational, 143: 153-156, 2004). Kits were purchased from IDS (St.Joseph, Mich.).

Surgery: All procedures, including the mandibular bleed, are conductedunder a standard protocol, MO05M56 (Section 3, Protocols), approved bythe Johns Hopkins Animal Care and Use Committee. Surgery is performedwith sterile equipment and aseptic conditions. Approximately 20 g miceare anesthetized with 0.15 mL of a solution containing ketamine andxylazine. A surgical surface on the right flank is shaved and washedwith ethanol and Betadyne. A 4-5 mm incision is made through the skin. Adrug pellet is inserted into the exposed SC cavity. The skin is apposedand stapled. Animals are observed until conscious and moving normally asspecified in the standard protocol. They are returned to their cages. Atintervals described in Results, approximately 100 μL of blood iscollected by mandibular bleeds for drug analyses and/or toxicity tests.

Clinical Chemistry: Samples of blood were analyzed for hematology andliver chemistry. Blood samples were collected 4-5 days before surgery toprovide baseline control values. Tests were performed by the MousePhenotyping Core of the Johns Hopkins University School of MedicineDepartment of Molecular and Comparative Pathobiology. A liver panelmeasures total protein, albumin, glucose, and the enzymes alkalinephosphatase, alanine aminotransferase, and aspartate aminotransferase.The hematology panel included platelets, hemoglobin, red and white cellindices, and white cell differential.

Pain Assessments: Pain was measured using the revised ethnogramdescribed by Krugner-Higby et al (2003) for measuring post surgical painin rats (Krugner-Higby, et al. Comparative Medicine 53: 270-279, 2003).Animals were observed daily for behavior, hair coat, eye appearance, andporphyrin staining.

Weight: Mice were weighed before and after implant using a Mettlerportable laboratory balance with a 0.01 gram read out.

Results

In vitro dissolution studies of the 5 mg pellets containing 1.0 mgconcentration of buprenorphine are shown in Table 1. Results areexpressed as the average of 3-4 samples for each sustained-releaseproduct. For comparison, the dissolution of 5 mg pellets of SABER (Okumuet al. Biomaterials 23 (22): 4353-4358, 2002.) and PCPP:SA(lactic-co-glycolic) acid copolymer (Shah et al., J Control Rel. 27(2):139-147, 1993) were also examined, each containing 1 mg of buprenorphinein the 5 mg pellet.

The in vitro data shown in Table 1 confirm the in vivo studies thatcholesterol pellets release buprenorphine at a constant rate. Forcomparison release studies with SABER and PCPP:SA biodegradablescaffolds show similar dissolution rates. The three sustained-releasescaffolds provided buprenorphine at approximately first-order releasekinetics. At least 50% of the dose remains in the animal 7-days afterimplant.

TABLE 1 Cumulative Buprenorphine Release at 37° in PBS from 5 mgCholesterol Pellet, PCPP:SA, and SABER Pellets Day 1 Day 3 Day 5 Day 7Scaffold % Drug Released Cholesterol Pellet (20% w:w) 13 26 36 49 PCPPSA10 22 34 50 SABER-15% EtOH 12 29 40 49

In vivo studies were conducted with 5 mg cholesterol pellets containing1.0 mg of buprenorphine to investigate the safety in mice of a nearlethal dose of buprenorphine (50 mg/kg). In the first set of tests, 9mice were implanted with 5 mg pellets. Mice were observed daily for thefirst week and at 2-3 day intervals thereafter, and were bled on days-1,2, 4, 7, 14, and 22.

One mouse died during bleeding on day-4. A second mouse died duringbleeding on day-7. Autopsy reports suggest the cause of death was bloodloss and spinal cord trauma due to excess pressure on the back of themouse as the technician attempted to grab the mouse for bleeding. Therewas no evidence of pathology associated with the implants. The remaininganimals were unremarkable. The animals were sitting in normal position,moving freely, grooming, bright and alert. Hair coat was normal. Theeyes were open and alert. There was no evidence of porphyrin stainingaround the eyes or nose. There was no change in weights compared to twocontrol mice that had surgery but no implants. The animals wereeuthanized 2 months after implant.

In a second safety study, eight mice were dosed with 5 mg pelletscontaining 1 mg of drug prepared as described above. Two animals werecontrols. Animals were observed daily for the first week and at 2-3 dayintervals thereafter. There was no remarkable change in behavior. Animalweights at day-0 and day-5 were the same. Because the two deaths in Test1 were suspected to be related to excessive bleeding (autopsy report),we limited blood collections to ca 100 μL/48 hrs. The zero-time samplesreported below represent samples of blood drawn 2-4 days before surgery.

Five mice (4 test, 1 control) were bled on days-0, 1, 3, 5, 7, and 14for hematology. Five mice (4 test, 1 control) were bled on days-0, 1, 3,5, 7, 14, 31, and 38 for liver function tests. Hematology tests wereconducted to establish baseline values and to rule out effects of theprocedure on sensitive bone marrow metabolism. Liver function tests wereselected because the drug is metabolized in the liver.

Hematology parameters were largely normal. By day-5, mice began toexhibit increased white and red blood cell counts, a well-known effectin sequentially bled mice. Liver function parameters were largelynormal. Alkaline phosphatase values were consistently, but slightly, outof range in test and control animals including the day-0 samples. Torule out hepatotoxicity, the liver function tests were extended today-38. There was no significant difference between day-0 and day-38values.

In summary, the results of the behavioral studies, weight, and bloodtests showed no difference between animals dosed with a 1 mg ofbuprenorphine and control animals.

To investigate whether in vivo release kinetics would be similar to thein vitro data shown in Table 1, blood samples were analyzed forbuprenorphine using an ELISA assay. The results in Table 2 show entirelyunexpected results. In contrast to the in vitro dissolution tests inTable 1 and to results reported in rats with 50 mg cholesterol pelletscontaining 10 mg of drug there is a hyperbolic release of drug on day-1to day-2 followed by a linear decline to day-14. By day-22 blood levelsfall below the level of detection.

TABLE 2 Plasma Buprenorphine with Chronic Delivery of 1 mg Buprenorphinein Cholesterol/Triglyceride Pellet Plasma Buprenorphine (ng/mL ± SD)Post-Delivery Day 1 2 4 7 14 22 140 ± 29 120 ± 28 16 ± 8 11 ± 7 9 ± 50.2 ± 0.2 * n = 4 for day-0; 9 for days 1-4; 7 for days 7 to 22 for day

The results shown in Table 2 were confirmed in a second set of bloodassays using samples of blood remaining after the hematology and liverfunction tests in the second safety study were measured for drug.Sufficient blood was not available from day-1 samples and for only abouthalf of the remaining samples. Nonetheless, the results shown in Table 3demonstrate that the in vivo release profile was similar to thebioavailability seen in Table 2.

TABLE 3 Repeat Plasma Buprenorphine with Chronic Delivery of 1 mgBuprenorphine in Cholesterol/Triglyceride Pellet Plasma Buprenorphine(ng/mL ± SD)* Post Delivery Day 1 3 5 7 31 ** 24 ± 24 13 ± 14 7.2 ± 1.60.0 n = 2 for day-0, 2 for day-3, 5 for days 5 and 7; ** no sampleavailable for test

To determine whether the hyperbolic release kinetics seen in the in vivostudies using pellets with 1 mg of drug would be observed in studieswith a cholesterol pellet containing a therapeutic level ofbuprenorphine, eight mice were dosed with a 5 mg pellet containing 0.1mg of drug. Two animals were controls. The results, shown in Table 4demonstrate that the proposed BPP affords clinically relevant plasmalevels of buprenorphine for up to 72 hours.

TABLE 4 Plasma Buprenorphine with Chronic Delivery of 0.1 mgBuprenorphine in Cholesterol/Triglyceride Pellets Plasma Buprenorphine(ng/mL ± SD)* Post-Delivery Day 1 3 5 9 16 ± 16 10 ± 14 1.5 ± 2.5 0

TABLE 5 Plasma Buprenorphine with Chronic Delivery of 0.1 mgBuprenorphine in Cholesterol/Triglyceride Pellets Prepared withoutSolvents and in Oleic Acid Emulsions Plasma Buprenorphine (ng/mL ± SD, n= 8) Post-Delivery Day 1 2 4 No Solvents 6 ± 2 0.5 ± 2 0 Oleic Acid 2 ±1 0 0

Cholesterol pellets were prepared without solvents by mixing 90 mgcholesterol, 8 mg triglyceride, and 2 mg buprenorphine for 48 hours at5° on a roller wheel. Eight 1-2 mg samples were collected randomly andassayed for buprenorphine to verify the homogeneity of the mixture. Fivemg pellets were pressed and implanted in 8 mice. The blood levels ofbuprenorphine at days 1-4 are shown in Table 5. Cholesterol pelletsprepared without chloroform-ethanol solution had significantly lesscapacity to provide sustained release analgesia. By day-2, blood levelsof drug on average were less than 0.5 ng/per mL. Oleic acid emulsions ofdrug injected SC afforded a similar failure to provide sustained-releaseanalgesia. The data in Table 5 shows that blood levels of drug from the50 microliter injections were 3-fold less compared to the solvent-freecholesterol pellet implant. There was no detectable buprenorphine atday-2.

Discussion

Buprenorphine arguably is the least likely analgesic drug to interferewith animal research. The work of Martin and colleagues in 1976 onchronic spinal injury in dogs confirmed the drug's action as a partialagonist at the mu-opioid receptor. Interest in the drug for more than 30years relates to its unique pharmacology, including low toxicity, highaffinity to and slow release from mu-opioid receptors, and dose-responsecurve in rodents [Flecknell, Lab Animal. 18: 147-160; 1984; Cowan, Int JClin Pract Suppl. February: 3-8, discussion 23-24; 2003].

Buprenorphine esters can act as buprenorphine pro drugs in sesame,castor, cottonseed, peanut or soybean oil solutions injected into ratmuscle (IM). The analgesic effect for sesame oil solutions could bemeasured for approximately 3 days (Liu, et al. Anesth Analg. 102:1445-1451, 2006; J Pharm Pharmacol. 58(3): 337-34, 2006). The laterstudies contrast with the inability described herein (Table 5) to detectbuprenorphine in blood following SC injections of buprenorphine in oleicacid, an oil with properties highly similar to the oils used by Liu andcoworkers.

The studies described herein demonstrate that short term release ofbuprenorphine from cholesterol-triglyceride implants is safe. Safety isclearly demonstrated via clinical chemistry studies of liver enzymes. Inall species tested, the drug is metabolized in the liver. The primarymetabolite is norbuprenorphine. Pharmacokinetic studies have beenconducted in rats. After bolus intravenous administration, plasma levelsdecline triexponentially. Unmetabolized drug excreted in the urine andfeces one week after injection was 1.9 and 22.4% of the dose,respectively, and 92% of the dose was accounted for in one week (PontaniXenobiotica, 15, 287-297, 1985). The plasma half-lives for buprenorphineand norbuprenorphine are about 4-5 hours (Gopal, et al. Eur JPharmaceutical Sciences, 15: 287-293, 2002). The LD₅₀ for a bolus IPdose of buprenorphine in mice and rats is ca 95 and 200 mg/kg,respectively (Cowan, et al. Br J Pharmacol, 60: 547-554, 1977). In thepresent study, near-lethal doses of drug given viacholesterol-triglyceride implant, produced no signs of toxicity. Dosesof 1 mg of drug, 50 mg/kg, had no effect on behavior or on sensitiveliver enzymes that are involved in drug metabolism.

The unexpected and surprising difference in drug availability from 50 mgand 5 mg cholesterol-triglyceride implants is illustrated in FIG. 1.FIG. 1 shows the amount of drug retained at the implant site from 50 mgversus 5 mg pellet implants. The data for the 50 mg implant is takenfrom the report of Pontani and Misra (1983) using their in vivo data andtheir formula for the first-order release kinetics of 10 mg ofbuprenorphine from their 50 mg pellet: “Exp 50 mg pellet/10 mg drug.”The plot of the in vitro data with 5 mg pellets containing 1 mg of drug(Table 1) is shown in FIG. 1 for comparison. The in vitro data with 5 mgpellets, “Exp 5 mg pellet/1 mg drug,” closely matches the Pontani andMisra data.

Therefore, it was expected that while 5 mg pellets may afford somelong-term possibilities for analgesia, the animals still would require 1to 2 days of post-surgical injections at 6-8 hour intervals. Moreover,these 50 mg and/or 5 mg analgesic pellets could not be used in manyresearch projects because the remaining somatic drug-load couldinterfere with metabolic and nerve studies. For example: the in vitrodata argues that a 5 mg pellet with 1 mg of buprenorphine would hold 50%of the dose at day-7, or 25% of the lethal dose for mice.

As shown in FIG. 1, the observed in vivo data was strikingly differentthan expected. The 5 mg implants with 1.0 and 0.1 mg of drug (data fromTable 2 and 4, respectively) had their maximum drug release in day-1 today-2. Following this burst analgesia, the animals get the maximum paintherapy in the 24 hours after surgery. Moreover, there is littledetectable drug remaining at day-5 to day-7. Thus, there is littleconcern that the analgesia can interfere with research objectives inlaboratory experiments.

The unexpected and surprising difference in drug availability from 50 mgand 5 mg cholesterol-triglyceride implants remains unexplained. While itis likely that the 3 mm by 1 mm long 5 mg pellet would erode faster thanthe 3 mm by 6 mm long 50 mg pellet described by Pontani and Misra(1983), one would expect similar release kinetics because in vitro testswith 5 mg pellets showed a release profile (Table 1) comparable to the50 mg pellet in vivo. It is not believed that the hardness of the pelletplayed a role. Optimal pressure was used to prepare the pellets becausemore force and longer compaction prevented uniform pellet formation,possibly due to thermal effects on the lipid matrix. Also, given thesame composition and production pressure, a smaller pellet should beharder and dissolve more slowly. If size and shape were the determiningfactor, one would expect to see the most rapid release withbuprenorphine in oleic acid oil. Yet, little or no drug was detected inplasma one day after an implant with oleic acid, although based on the 5hr half-life of the drug in plasma, at least 12.5 μg of buprenorphineshould be available. The remarkably different blood levels of drugobserved from cholesterol-triglyceride-drug implants prepared by thesolvent-evaporation method and implants prepared by blending ingredientswithout solvent (Table 5) suggest that buprenorphine and/or thetriglyceride in organic solvent affords a unique particle.

One concern about the 50 mg implant is that it takes 12 or more weeks todissolve in vivo. A second concern is that in vivo tests demonstratefirst order kinetics for drug release. The slow linear release of drugmeans that the animal still would need several bolus injections ofbuprenorphine within the 24-hour period after surgery when pain isgreatest.

In vitro dissolution tests demonstrated a linear drug release profilefor the 5 mg pellet. It is logical to anticipate that it would have thesame in vivo release deficiencies for analgesia as the 50 mg implant.However, in vivo mouse studies demonstrated entirely different andunexpected results. The 5 mg implants containing 0.1 mg of drug micedissolved in approximately 3 days. Moreover, the in vivo studiesdemonstrated highly desirable burst kinetics. Plasma levels of drugpeaked at day-1, a drug level that obviates the need for bolusinjections of analgesia immediately after surgery.

While the surprising difference between the in vivo and in vitro resultsremains unexplained, the data unequivocally demonstrate that 5 mgimplants can be used safely, and the 5 mg implants provide effectiveshort-term analgesia for mouse surgery. Moreover, the product solves thechallenge of providing post-surgical analgesia to small animals, aproblem that has been known for more than 25 years. The 5 mg implant forthe first time provides medical scientists with an easy-to-use, safetool to meet international animal welfare regulations and nationalguidelines.

I claim:
 1. A formulation for administration of an effective amount ofdrug to an animal over a period of between two days and nine days,consisting essentially of A dispersion of a drug uniformly distributedin a lipophilic carrier consisting of a sterol and fatty acid ester in aloading of 0.1 to 50% by weight, wherein the formulation releases drugselected from the group consisting of local anesthetics, opioids,fentanyl, buprenorphine, and butorphanol to provide clinicallysignificant levels of drug over a period of between two and nine daysand release of drug is complete over a period of between two andfourteen days.
 2. The formulation of claim 1 wherein the carrier is amixture of cholesterol and fatty acid ester.
 3. The formulation of claim1 wherein the drug is added to between 0.1 and 50% by weight.
 4. Theformulation of claim 1 in a single use syringe.
 5. A method of making aformulation for administration of an effective amount of drug to ananimal over a period of between two days and nine days, comprisingproviding a solution of a lipophilic carrier consisting of a sterol andfatty acid ester in a solvent, mixing uniformly into the carrier a drugselected from the group consisting of local anesthetics, opioids,fentanyl, buprenorphine, and butorphanol in a loading of 0.1 to 50% byweight, removing the solvent from the mixture to yield drug-carrierparticles, and forming a dispersion of the drug-carrier particles.
 6. Amethod of providing drug to an animal comprising administering to theanimal an effective amount of a formulation to provide an effectiveamount of drug over a period of between two days and nine days, theformulation comprising A dispersion of drug-carrier particles consistingessentially of drug uniformly distributed in a lipophilic carrierconsisting of a sterol and fatty acid ester in a loading of 0.1 to 50%by weight, wherein the formulation releases drug at a constant rate toprovide clinically significant levels of drug over a period of betweentwo and nine days and release of drug is complete by fourteen days. 7.The method of claim 6 wherein the formulation is administered at surgerytime or by implantation or injection through a catheter or trocar. 8.The method of claim 7 wherein the formulation is administeredintramuscularly, subcutaneously, or intraperitoneally.
 9. Theformulation of claim 1 wherein all of the drug is released over a periodof two to nine days.
 10. The formulation of claim 1 wherein the carrierconsists of cholesterol and fatty acid ester and the drug is selectedfrom the group consisting of local anesthetics, opioids, fentanyl,buprenorphine, and butorphanol.
 11. The formulation of claim 9 whereinthe carrier consists of cholesterol and fatty acid ester and the drug isbuprenorphine.
 12. The method of claim 5 wherein all of the drug isreleased over a period of two to nine days.
 13. The method of claim 5wherein the carrier consists of cholesterol and fatty acid ester and thedrug is selected from the group consisting of local anesthetics,opioids, fentanyl, buprenorphine, and butorphanol.
 14. The method ofclaim 13 wherein the carrier consists of cholesterol and fatty acidester and the drug is buprenorphine.
 15. The method of claim 6 whereinall of the drug is released over a period of two to nine days.
 16. Themethod of claim 6 wherein the carrier consists of cholesterol and fattyacid ester and the drug is selected from the group consisting of localanesthetics, opioids, fentanyl, buprenorphine, and butorphanol.
 17. Themethod of claim 16 wherein the carrier consists of cholesterol and fattyacid ester and the drug is buprenorphine.